Project Summary Molecular-scale interactions lie at the heart of cell biology, and super-resolution microscopy has emerged as a powerful tool for measurements inside cells. However, the current state-of-the-art does not include a generalizable method for mapping, measuring, and understanding real-time motions and biochemical interactions of pairs of mobile, dynamic components within cells. This gap is particularly critical in bacteria cells, in which many microscopy technologies are unproven or unfeasible due to small size and impenetrable walls. Something fundamentally different is needed: a new approach to understanding subcellular biochemistry in cells in real time. Thus, we aim to bridge single-molecule tracking, super-resolution imaging, and probabilistic statistical modeling to study pairs of molecules with the long-term goal of mapping the biochemistry of subcellular components in living bacteria cells. This exploratory proposal will develop interaction maps in living bacteria cells with two specific aims: (1) To observe pairs of mobile proteins and genetic loci with fluorescent protein fusions, CRISPR-dCas9 labeling, and super-resolution fluorescence microscopy in living bacteria; and (2) To measure the dynamics and interactions of these subcellular pairs with single-molecule tracking, Bayesian inference, and statistical modeling. The tools developed in this project will immediately benefit single-molecule imaging research by increasing the attainable information content. Furthermore, by focusing on applications in bacterial cell biology, the tools developed in this project will have a widespread, positive biomedical impact by making accessible long-term, significant questions in microbial biology.